[A first pilot study on the neonatal screening of primary immunodeficiencies in Spain: TRECS and KRECS identify severe T- and B-cell lymphopenia]. / Primer estudio piloto en España sobre el cribado neonatal de las inmunodeficiencias primarias: TRECS y KRECS identifican linfopenias T y B graves.

INTRODUCTION: Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected infants/children. The measurement of T-cell receptor excision circles (TRECS) and kappa-deleting recombination excision circles (KRECS) can identify neonates with severe T or B-cell lymphopenia. OBJECTIVES: To determine TRECS and KRECS levels from prospectively collected dried blood spot samples (DBS) and to correctly identify severe T and B-cell lymphopenia. MATERIAL AND METHODS: Determination of TRECS and KRECS by multiplex PCR from neonates born in two tertiary hospitals in Seville between February 2014 and May 2014. PCR cut-off levels: TRECS<15 copies/µl, KRECS<10 copies/µl, ACTB (ß-actin)>1000 copies/µl. Internal (XLA, ataxia telangiectasia) and external (SCID) controls were included. RESULTS: A total of 1068 out of 1088 neonates (mean GA 39 weeks (38-40) and BW 3238g (2930-3520) were enrolled in the study. Mean (median, min/max) copies/µl, were as follows: TRECS 145 (132, 8/503), KRECS 82 (71, 7/381), and ACTB 2838 (2763, 284/7710). Twenty samples (1.87%) were insufficient. Resampling was needed in one neonate (0.09%), subsequently giving a normal result. When using lower cut-offs (TRECS<8 and KRECS<4 copies/µl), all the samples tested were normal and the internal and external controls were correctly identified. CONCLUSION: This is the first prospective pilot study in Spain using TRECS/KRECS/ACTB-assay, describing the experience and applicability of this method to identify severe lymphopenias. The ideal cut-off remains to be established in our population. Quality of sampling, storage and preparation need to be further improved.

HCV RNA in peripheral blood cell subsets in HCV-HIV coinfected patients at the end of PegIFN/RBV treatment is associated with virologic relapse.

Extrahepatic replication may have important implications for the treatment of hepatitis C virus (HCV). Our aim was to analyse the association between the presence of positive/negative strand HCV RNA in different peripheral blood cell subsets at the end of PegIFN/RBV treatment, and treatment response in HIV-coinfected patients. Thirty-four HCV-HIV coinfected patients who concluded 48 weeks of PegIFN/RBV treatment were included in the present study. Positive/negative strand HCV RNA was detected by amplification of the 5' untranslated region (5' UTR) using high-temperature RT-PCR in immunomagnetic-isolated cell subsets. Twenty-three patients (67.6%) had sustained virologic response (SVR) while 11 patients (32.4%) relapsed. The frequency of positive/negative strand HCV RNA in any cell subsets was significantly lower in patients with SVR (8.6%) compared to relapsers (63.6%) (P = 0.002). Baseline HCV viral load was statistically higher among patients who relapsed (P = 0.008), while patients with SVR had very early virologic response more frequently (P = 0.003). Multivariate analysis showed, among these three variables, that only the presence of positive/negative strand HCV RNA was independently associated with relapse [P = 0.024; OR 14 (14-137)]. In conclusion, the presence of positive/negative strand HCV RNA at the end of treatment is associated with relapse among HCV-HIV coinfected patients and might have important implications in the clinical practice.

Effect of the substitution of one nucleoside analogue by one non-nucleoside reverse transcriptase inhibitor over mitochondrial DNA levels.

BACKGROUND: Long-term antiretroviral therapy is associated with several side effects, like mitochondrial toxicity related to nucleoside reverse transcriptase inhibitors (NRTIs). Our objective was to analyze the effect of the substitution of one NRTI by one non-nucleoside reverse transcriptase inhibitor (NNRTI) in the antiretroviral regime of HIV-1-infected patients who were on a regime containing either two NRTIs and one NNRTI, or one NRTI, one NNRTI and one protease inhibitor (PI), over mtDNA level. Decreasing NRTIs could increase mtDNA level. METHODS: Fifteen HIV-1-infected patients were included in the study. As controls, 17 healthy individuals and 15 HIV-1-infected patients naïve for antiretroviral treatment were also analyzed. mtDNA level was quantified at baseline and after 48 weeks of treatment. RESULTS: Control groups showed higher levels of mtDNA than the study group (p<0.001). Among this latter group, no statistical differences between baseline and after 48 weeks were found. Naïve HIV-infected patients had lower mtDNA than healthy volunteers (p<0.001). Two patients had two consecutive blips (low viral load increases) but they did not show NNRTI-related resistance mutations. CONCLUSIONS: This study shows that although this treatment was immunovirologically effective, mtDNA level did not increase at least after 48 weeks.